Method for detecting streptococcus agalactiae using esterase activity

ABSTRACT

A reaction medium that includes (i) an esterase enzyme substrate that  Streptococcus agalactiae  is not capable of using at less than 18 hours after inoculation, (ii) at least one enzymatic substrate selected from β-cellobiosidase substrates, N-acetylglucosaminidase substrates, and a β-glucosidase substrates, and (iii) a phosphatase substrate.

This is a divisional of application Ser. No. 11/660,386 filed Feb. 16,2007, which is a National Stage Application of PCT/FR2005/050739 filedSep. 14, 2005, and claims the benefit of French Application No. 04.52068filed Sep. 16, 2004. The entire disclosures of the prior applicationsare hereby incorporated by reference in their entirety.

The present invention relates to the field of the detection andidentification of Streptococcus agalactiae. More specifically, theinvention relates to the use of esterase substrates, optionally incombination with at least one α-glucosidase substrate, phosphatasesubstrate, β-cellobiosidase substrate or N-acetylglucosaminidasesubstrate, for detecting and identifying Streptococcus agalactiae.

The Streptococcus genus contains numerous species that are verywidespread in nature, on the skin and the mucous membranes of humans andanimals, and are responsible for multiple infections. They areubiquitous bacteria that are found in the free state in the outsideenvironment (soil, air, water), in the saprophyte state or in thecommensal state in humans and animals. They are located in therhinopharynx for group A, C, G and H streptococci and salivarius, theintestine for group D fecal streptococci and the vaginal cavity forgroup B streptococci. Their pathogenic role is extremely varied anddepends on the species in question and on their location in theorganism.

Streptococci are Gram+cocci, 0.5 to 1 μm in diameter, that exhibitgrouping in the form of a small chain and are immobile. They arecatalase-negative, have a fermentative metabolism, and they areoptionally anaerobic and are sensitive to variations in temperature(optimal growth 37° C.) and to variations in pH (optimal pH 7).

Streptococcus agalactiae (or streptococcus B) is recognized as one ofthe main infectious agents responsible for mastitis in cattle. Inhumans, it is essentially a saprophyte of the female genital tract(vagina), but it is also found in the rhinopharynx and in the intestine,in particular the rectum. In adults, colonization often remainsasymptomatic, but Streptococcus agalactiae can be responsible forsepticemia, pneumonia, meningitis, arthritis, urinary infections anddeep suppurations. In women who are pregnant, or after having givenbirth, the infection may lead to endometritis and to sterility.

In newborns, the contamination occurs in utero or, most commonly, duringbirth, due to inhalation of the amniotic fluid or of vaginal secretions.An early infection often appears immediately after birth or within thefirst hours of life. Early infection is promoted by premature birth,rupture of the membranes and a strong colonization of the mother'svagina. The mortality rate in this type of infection is very high(>50%). Late infections are generally reflected by meningitis (infantilemeningitis) and arthritis.

Systematic screening for the carrying of Streptococcus agalactiae isrecommended at the end of pregnancy, ideally between 34 and 38 weeks ofamenorrhea (35-37 weeks of pregnancy), due in particular to itsprevalence (10% in France, i.e. at least 75 000 pregnant women/year) andto the consequences thereof during full-term births, which makes it apublic health problem.

Selective media and/or media which make it possible to direct thediagnosis are commercially available. However, these media have thedrawback that they are not sufficient on their own for the diagnosis ofStreptococcus agalactiae and that it is necessary to carry outsupplementary tests, such as demonstrating group B Lancefield antigen(polysaccharide with dominant presence of rhamnose) and hippuratehydrolysis (hippurate broth).

The selective media most commonly used are Todd-Hewitt broth, anenrichment broth for searching for group B streptococci in pregnantwomen. This broth contains various antibiotics that inhibit mostGram-negative microorganisms of the accompanying flora, such asnalixidic acid and gentamycin, or nalixidic acid, polymyxin and crystalviolet.

After the enrichment step, the antibiotic-supplemented Todd-Hewitt brothmust be subcultured on media for searching for streptococci (see CDC(Center for Disease Control) recommendations, MMWR (Morbidity andMortality Weekly Report), 16 Aug. 2002, Vol. 51, No. RR-11).

Lim medium is a variant of Todd-Hewitt broth and it contains 1% of yeastextract, nalixidic acid and colistin.

A Columbia agar containing 5% of blood is also used and makes itpossible in particular to demonstrate the β-hemolytic characteristic ofStreptococcus agalactiae. However, this characteristic is not alwaysapparent: the hemolytic halo around the colonies may be narrow, givingrather the α-hemolytic, or even γ-hemolytic, appearance. On the otherhand, this characteristic becomes clear if, in the area of theStreptococcus agalactiae colonies, there are Staphylococcus aureuscolonies (Camp-factor).

The drawbacks of these selective media are that they must besupplemented with biochemical tests and/or immunoassays.

Currently, the only commercially available, ready-to-use selectivemedium that makes it possible to directly isolate and identifyStreptococcus agalactiae from rectovaginal samples is Granada medium(Biolys SA). This medium has the characteristic of promoting theproduction of a carotenoid pigment by Streptococcus agalactiae strainsdue to the presence in the medium of soluble starch, proteose peptoneNo. 3, glucose, sodium pyruvate, magnesium sulfate, methotrexate,colistin, crystal violet, agar, horse serum, anhydrous Na₂HPO₄,metronidazole, MOPS (morpholinopropanesulfonic acid) hemi-sodium saltand distilled water, and incubation under anaerobic conditions. Thismedium therefore has the drawback that the direct detection ofStreptococcus agalactiae is carried out under anaerobic conditions,which is not easy to implement. Moreover, no detection medium containingone or more enzymatic substrates is available.

The applicant has now demonstrated, against all expectations, that it ispossible to use enzymatic substrates, in particular esterase enzymaticsubstrates, for specifically detecting and identifying Streptococcusagalactiae.

Specifically, surprisingly, the applicant has demonstrated that onlyStreptococcus agalactiae, among the closest bacterial species mostcommonly encountered in an associated manner, are incapable of usingesterase enzymatic substrates early on (at less than 18 h afterinoculation), such that they are the only ones not to be revealed earlyon by esterase substrates, for example with no modification of thecolonies being obtained in the medium early on, for example with nomodification of the coloration of the colonies being obtained in themedium when a chromogenic esterase substrate is used, without therebeing any diffusion of the coloration in the reaction medium, andtherefore with the coloration being concentrated at the colonies,without these molecules having, however, a harmful effect on the growthof the bacteria.

Consequently, this enzymatic substrate has the additional advantage thatthe results can be read early, in particular at approximately 18-20 h ofincubation, with a very good contrast.

Thus, a subject of the present invention is a method for specificallydetecting and identifying Streptococcus agalactiae, characterized inthat a reaction medium comprising at least one esterase enzymaticsubstrate is used.

The esterase enzymatic substrates that are suitable for the purposes ofthe invention are any substrate known to those skilled in the art thatmakes it possible to demonstrate such an enzymatic activity. Suchsubstrates may, for example, be chromogenic or fluorescent and aredescribed, for example, in the BIOSYNTH catalog, Substrates and Reagentsor www.biosynth.com, or in the GLYCOSYNTH catalog, enzyme substratescatalog or www.glycosynth.co.uk.

By way of example of esterase substrates, mention may be made ofindoxyloctanoate, indoxylnonanoate or indoxyldecanoate derivatives,preferably indoxyloctanoate derivatives, more preferably theirhalogenated derivatives, more preferably the chlorinated or brominatedderivatives such as 5-bromo-6-chloro-3-indoxyloctanoate and5-bromo-4-chloro-3-indoxyloctanoate for which the reading isparticularly early.

Since a slight esterase activity is observed after 24 h of incubation(activity less than 0.6 on a scale of 0 to 4), the detection ofStreptococcus agalactiae can be improved by adding at least one otherenzymatic substrate. Due to the specific property of Streptococcusagalactiae of not using the esterase substrate, or of using it verylittle, it makes no difference whether or not the other enzymaticsubstrate is used by Streptococcus agalactiae and the other species. Inaddition, since the use of the esterase substrate by Streptococcusagalactiae is only very slight, such that this only slightly modifiesthe appearance of the colonies obtained, we will indicate, in thesubsequent text, only that Streptococcus agalactiae are incapable ofusing the esterase substrate.

Thus, according to one embodiment, the method of the invention uses areaction medium also comprising another enzymatic substrate other thanan esterase substrate.

The enzymatic substrates other than an esterase substrate (non-esterasesubstrate) suitable for the purposes of the invention are any substrateof which the use by a strain confers on the colony an appearancedifferent than the appearance obtained when the esterase substrate isused. Such a different appearance is, for example, a differentcoloration. Moreover, this non-esterase substrate is such that, when astrain uses both this non-esterase substrate and the esterase substrate(strain other than Streptococcus agalactiae), the appearance of thecolonies obtained (for example, their coloration) is also different thanthe appearance of the colonies of Streptococcus agalactiae.Specifically, when both an esterase substrate and a non-esterasesubstrate that can be used by the Streptococcus agalactiae strains arecombined in a reaction medium, the Streptococcus agalactiae strains arethen negative for the esterase and positive for the non-esterasesubstrate (they can be marked −/+, the first part of the equationcorresponding to the esterase substrate and the second partcorresponding to the non-esterase substrate), while the other strainsare capable of using either only the esterase substrate (they are +/−),or both the esterase substrate and the non-esterase substrate (they are+/+). Similarly, when both an esterase substrate and a non-esterasesubstrate that cannot be used by the Streptococcus agalactiae strainsare combined in a reaction medium, the Streptococcus agalactiae strainsare then negative for the esterase and negative for the non-esterasesubstrate (they are −/−), while the other strains are capable of usingeither only the esterase substrate (they are +/−), or both the esterasesubstrate and the non-esterase substrate (they are +/+). In summary, theStreptococcus agalactiae strains are always −/+ or −/−, whereas theother species are always +/− or +/+.

Thus, for example, if a chromogenic esterase substrate which results ina blue coloration of the colonies when the colony under considerationuses the substrate is combined with another chromogenic enzymaticsubstrate which results in a pink coloration of the colonies when thecolony under consideration uses the substrate, four types of colorationcan be obtained: either pink, or colorless to slightly blue, or blue, orviolet (pink+blue). The pink coloration and the colorless to slightlyblue appearance are only representative of Streptococcus agalactiae asfollows: either the strain is capable of using the non-esterasesubstrate and the colony becomes pink (−/+ strain), or it is incapableof using the non-esterase substrate and the colony remains colorless orbecomes slightly blue (−/− strain). The blue and violet colorations arerepresentative of the other species as follows: either the strain iscapable only of using the esterase substrate and it becomes blue (+/−strain), or the strain is capable of using both the esterase substrateand the other enzymatic substrate and it becomes pink and blue, i.e.

violet (+/+ strain).

Similarly, if a fluorescence-absorbing esterase substrate, which resultsin quenching of fluorescence when the colony under consideration usesthe substrate, is combined with another fluorescent enzymatic substratewhich results in fluorescence at the colonies when the colony underconsideration uses the substrate, the latter substrate being used byStreptococcus agalactiae, two types of colonies can be obtained: eitherfluorescent colonies, or weakly to non-fluorescent colonies. Thefluorescent colonies are representative only of Streptococcus agalactiaesince this species is only capable of using the enzymatic substrateother than the esterase substrate. The weakly to non-fluorescentcolonies are representative of the other species as follows: either thestrain is only capable of using the esterase substrate and it isnon-fluorescent, or the strain is capable of using both the esterasesubstrate and the other enzymatic substrate and it is weakly tonon-fluorescent.

Examples of such substrates other than an esterase substrate that aresuitable for the purposes of the invention include α-glucosidasesubstrates, phosphatase substrates, β-cellobiosidase substrates,N-acetylglucosaminidase substrates and β-glucosidase substrates.

Thus, according to another embodiment, the method of the invention uses,as reaction medium, a reaction medium comprising, in addition to anesterase substrate, at least one enzymatic substrate chosen fromα-glucosidase substrates, phosphatase substrates, β-cellobiosidasesubstrates, N-acetylglucosaminidase substrates and β-glucosidasesubstrates.

The reaction media comprising or consisting of an esterase substrate and(of) at least one enzymatic substrate chosen from an α-glucosidasesubstrate, a phosphatase substrate and a β-cellobiosidase substrate arenovel and constitute another subject of the invention.

The α-glucosidase enzymatic substrates suitable for the purposes of theinvention are any substrate known to those skilled in the art that makesit possible to demonstrate such an enzymatic activity. Such substratesmay, for example, be chromogenic or fluorescent and are described, forexample, in the BIOSYNTH catalog, Substrates and Reagents orwww.biosynth.com, or in the GLYCOSYNTH catalog, enzyme substratescatalog or www.glycosynth.co.uk.

By way of example of an α-glucosidase substrate, mention may be made ofindoxyl-derivative-based substrates, umbelliferone-derivative-basedsubstrates and naphthol-derivative-based substrates.

Preferably, the α-glucosidase enzymatic substrate suitable for thepurposes of the invention is an indoxyl-derivative-based substrate.

Examples of such indoxyl derivatives include derivatives of3-indolyl-α-D-glucopyranoside, preferably halogenated derivatives ofthese compounds. By way of examples of halogenated3-indolyl-α-D-glucopyranoside derivatives, mention may be made of6-bromo-3-indolyl-α-D-glucopyranoside,5-bromo-6-chloro-3-indolyl-α-D-glucopyranoside,5-bromo-4-chloro-3-indolyl-α-D-glucopyranoside,5-bromo-4-chloro-3-indolyl-N-methyl-α-D-glucopyranoside and6-chloro-3-indolyl-α-D-glucopyranoside, the latter compound beingparticularly preferred.

The phosphatase enzymatic substrates suitable for the purposes of theinvention are any substrate known to those skilled in the art that makesit possible to demonstrate such an enzymatic activity. Such substratesmay, for example, be chromogenic or fluorescent and are described, forexample, in the BIOSYNTH catalog, Substrates and Reagents orwww.biosynth.com.

By way of example of a phosphatase substrate, mention may be made ofindolyl-derivative-based substrates, umbelliferone-derivative-basedsubstrates and nitrophenyl-based substrates.

Preferably, the phosphatase enzymatic substrate suitable for thepurposes of the invention is an indoxyl-derivative-based substrate.

Examples of such indoxyl derivatives include 3-indolyl phosphatederivatives such as 5-bromo-4-chloro-3-indolyl phosphate,5-bromo-6-chloro-3-indolyl phosphate and 6-chloro-3-indolyl phosphate,the latter compound being particularly preferred.

The β-cellobiosidase enzymatic substrates suitable for the purposes ofthe invention are any substrate known to those skilled in the art thatmakes it possible to demonstrate such an enzymatic activity. Suchsubstrates may, for example, be chromogenic or fluorescent and aredescribed, for example, in the BIOSYNTH catalog, Substrates and Reagentsor www.biosynth.com or in the GLYCOSYNTH catalog, enzyme substratescatalog or www.glycosynth.co.uk.

By way of example of β-cellobiosidase substrate, mention may be made ofindolyl-derivative-based substrates, umbelliferone-derivative-basedsubstrates and nitrophenyl-based substrates.

Preferably, the β-cellobiosidase enzymatic substrate suitable for thepurposes of the invention is an indoxyl-derivative-based substrate.

Examples of such indoxyl derivatives include 3-indolyl-β-D-cellobiosidederivatives such as 6-chloro-3-indolyl-□-D-cellobioside and5-bromo-4-chloro-3-indolyl-β-D-cellobioside, the latter compound beingparticularly preferred.

The N-acetylglucosaminidase enzymatic substrates suitable for thepurposes of the invention are any substrate known to those skilled inthe art that makes it possible to demonstrate such an enzymaticactivity. Such substrates may, for example, be chromogenic orfluorescent and are described, for example, in the BIOSYNTH catalog,Substrates and Reagents or www.biosynth.com or in the GLYCOSYNTHcatalog, enzyme substrates catalog or www.glycosynth.co.uk.

By way of example of an N-acetylglucosaminidase substrate, mention maybe made of indoxyl-derivative-based substrates,umbelliferone-derivative-based substrates and nitrophenyl-basedsubstrates.

Preferably, the N-acetylglucosaminidase enzymatic substrate suitable forthe purposes of the invention is an indoxyl-derivative-based substrate.

Examples of such indoxyl derivatives include3-indolyl-β-N-acetyl-glucosaminide derivatives such as5-bromo-6-chloro-3-indolyl-N-acetyl-β-D-glucosaminide,6-chloro-3-indolyl-N-acetyl-β-D-glucosaminide and5-bromo-4-chloro-3-indolyl-β-N-acetylglucosaminide, the latter compoundbeing particularly preferred.

The β-glucosidase enzymatic substrates suitable for the purposes of theinvention are any substrate known to those skilled in the art that makesit possible to demonstrate such an enzymatic activity. Such substratesmay, for example, be chromogenic or fluorescent and are described, forexample, in the BIOSYNTH catalog, Substrates and Reagents orwww.biosynth.com or in the GLYCOSYNTH catalog, enzyme substrates catalogor www.glycosynth.co.uk.

By way of example of a β-glucosidase substrate, mention may be made ofindolyl-derivative-based substrates, umbelliferone-derivative-basedsubstrates and nitrophenyl-based substrates.

Preferably, the β-glucosidase enzymatic substrate suitable for thepurposes of the invention is an indoxyl-derivative-based substrate.

Examples of such indoxyl derivatives include3-indolyl-β-D-glucopyranoside derivatives such as5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside,5-bromo-6-chloro-3-indolyl-β-D-glucopyranoside,6-chloro-3-indolyl-β-D-glucopyranoside and5-bromo-4-chloro-3-indolyl-N-methyl-β-D-glucopyranoside.

According to one embodiment, the method of the invention uses a reactionmedium comprising i) an esterase substrate and ii) a phosphatasesubstrate or an α-glucosidase substrate, the esterasesubstrate/phosphatase substrate combination being preferred.

According to another embodiment, the reaction medium comprises, inaddition to the esterase substrate and the phosphatase substrate orα-glucosidase substrate, an enzymatic substrate chosen from aβ-cellobiosidase substrate, an N-acetylglucosaminidase substrate and aβ-glucosidase substrate, preferably a β-cellobiosidase substrate and anN-acetylglucosaminidase substrate.

The reaction medium as used in the method of the invention is thereforea detection reaction medium due to the presence of at least oneenzymatic substrate.

This reaction medium can either be used as a visualization medium only,or as a culture and visualization medium. In the first case, theculturing of the microorganisms is carried out before inoculation and,in the second case, the reaction medium also constitutes the culturemedium.

The reaction medium may be solid, semi-solid or liquid. The term “solidor semi-solid medium” is intended to mean, for example, a gelled medium.

Agar is the conventional solid medium in microbiology for culturingmicroorganisms, but it is possible to use gelatin or agarose. A certainnumber of preparations are commercially available, for instance Columbiaagar, trypcase-soy agar, MacConkey agar, Sabouraud agar or, moregenerally, those described in the Handbook of Microbiological Media (CRCPress).

The amount of agar in the reaction medium is from 2 to 40 g/l. For thesolid media, the amount of agar is preferably from 9 to 25 g/l, morepreferably from 12 to 14 g/l. For the semi-solid media, the amount ofagar is preferably from 2 to 6 g/1.

The enzymatic substrates of the invention can be used in a wide pHrange, in particular between pH 5.5 and 10.

The concentration of the enzymatic substrate(s) in the reaction mediumis between 10 and 2000 mg/l, preferably between 50 and 500 mg/l, morepreferably between 80 and 400 mg/l, which constitutes a preferredembodiment of the invention.

Of course, those skilled in the art will determine the concentration ofthe enzymatic substrate(s) in the medium within this range, according tothe substrate chosen. Thus, insofar as the esterase substrate used is5-bromo-4-chloro-3-indolyloctanoate, a concentration of between 100 and400 mg/l is preferred.

The reaction medium that can be used for the purposes of the inventionmay also comprise other components that are of use for improving thespecificity and/or the sensitivity of the method of the invention.

Thus, according to one embodiment of the invention, the reaction mediumcomprises phosphate solutions such as Na₂HPO₄ and K₂HPO₄ solutions.

This is because the use of such phosphate solutions makes it possible tosubstantially improve the readability of the medium, which is reflectedeither by a strength in coloration sharpness, or by an increase in theexpression and/or in the detection of the phosphatase activity at 18 h.

The concentration of such phosphate solutions is between 0.3 and 1.5 g/lfor each solution, a concentration of 0.5 g/l being preferred.

The reaction medium may also contain a mixture of inhibitors forinhibiting or limiting the growth of unwanted strains, such asfalse-positive strains, for example Candida or Staphylococcussaprophyticus, without modifying the detection sensitivity of themedium.

In this respect, the reaction mixture may contain a mixture ofantibiotics. The addition of antibiotics to the reaction medium allows,inter alia, a time to be saved since the identification of Streptococcusagalactiae is carried out directly.

Examples of antibiotics that are suitable for the purposes of theinvention include aztreonam and amphotericin B. These antibiotics arecommercially available from ICN, Squibb or Sigma.

The amount of each antibiotic in the reaction medium varies according tothe antibiotic concerned, and will be readily determined by thoseskilled in the art.

The reaction medium may also comprise one or more elements incombination, such as amino acids, peptones, carbohydrates, nucleotides,minerals, vitamins, surfactants, buffers, phosphate salts, ammoniumsalts, sodium salts or metal salts. Examples of media are described inthe applicant's patent applications EP 656 421 and WO 99/09207.

The implementation of the method of the invention can be carried outaccording to the following steps consisting in:

-   -   a) inoculating a reaction medium as defined above, with all or        part of the sample,    -   b) incubating the inoculated medium,    -   c) revealing the presence of at least one esterase activity        alone or in combination with at least one other enzymatic        activity other than an esterase activity,        which constitutes another subject of the invention.

The inoculation and incubation steps are widely known to those skilledin the art.

For example, the incubation temperature may be 37° C. As regards theincubation atmosphere, it is preferably aerobic.

The revealing is carried out with the naked eye by visualization of achange in coloration that does not diffuse in the reaction medium and istherefore concentrated at the colonies. In the case of the revealing ofthe fluorescence, the fluorescence reading devices known to thoseskilled in the art are used.

The biological samples to be analyzed are any clinical sample liable tocontain Streptococcus agalactiae, such as a vaginal specimen, a urinespecimen or any other sample of which the analysis may aid a clinicianin reaching a diagnosis.

The invention will be understood more clearly from the followingexamples given by way of nonlimiting illustration.

EXAMPLE 1 Detection of Streptococcus Agalactiae Using Esterase EnzymaticSubstrates 1.1 Preparation of the Reaction Media

The reaction media were prepared by mixing heart-brain extract (4.84g/l; Solabia), meat infusion (1.96 g/l; Solabia), biothione (1 g/l;Solabia), biotrypcase (7.2 g/l; Solabia), sodium carbonate (0.3 g/l;VWR), sodium pyruvate (2 g/l; Fluka), HEPES buffer (0.4 g/l; Sigma),lactalbumin peptone (2 g/l; DMV), glucose (1 g/l; Merck), American agar(2 g/l; Sobigel) and European agar (12 g/l; Roko).

After autoclaving for 15 min at 121° C., an esterase enzymatic substrateas indicated below was added at a rate of 0.3 g/l; followed by coolingin a water bath at 50° C.:

-   -   5-bromo-4-chloro-3-indolyloctanoate (X-C8; Inalco), which gives        a turquoise coloration when it is used, and    -   5-bromo-6-chloro-3-indolyloctanoate (Magenta-C8; Inalco), which        gives a pink-red coloration when it is used.

The media were then poured into a Petri dish for the subsequentinoculation with bacterial strains.

1.2 Inoculation of the Microorganism Strains

Three Streptococcus agalactiae strains and three strains of otherbacteria, all from the applicant's collection, suspended inphysiological saline, were inoculated so as to give isolated colonies oneach of the media. The dishes were incubated at 37° C. for 48 hours. Thecolonies formed were examined visually after 18, 24 and more than 40hours of incubation. The coloration of these colonies, the growth andalso the intensity of this coloration (representative of the esteraseactivity) were noted.

1.3 Results

The results are given in Table 1 hereinafter and are expressed:

-   -   in terms of growth (G) with the size being indicated in mm,    -   in terms of color (Co) with T=turquoise, R=pink or red,    -   in terms of intensity (I) of coloration, based on an arbitrary        scale ranging from 0 to 4, 0 corresponding to an absence of        activity and 4 corresponding to the presence of a very intense        coloration,    -   according to the incubation time in hours (T).

TABLE 1 Strains X-C8 Magenta-C8 (accession No.) T G Co I G Co IStreptococcus 18 1.2 1.2 agalactiae 24 2 T 0.3 2 R 0.3 (7611003) >40 2.5T 2.3 2.5 R 1.7 Streptococcus 18 0.4 0.4 agalactiae 24 0.7 T 0.3 0.7 R0.3 (0101060) >40 1.3 T 3 1.3 R 2 Streptococcus 18 0.3 agalactiae 24 0.20.5 (8904053) >40 0.3 T 0.3 1 Enterococcus 18 0.8 T 1.7 0.8 R 1 faecalis24 2 T 3 1.8 R 1.7 (0008192) >40 2 T 3.5 2 R 3.5 Enterococcus 18 0.5 T 20.7 R 1 faecium 24 1 T 3 1.7 R 2.7 (7611005) >40 1 T 3 1.7 R 3Staphylococcus 18 0.5 T 2 0.5 R 2 (7509009) >40 1.5 T 3 1.3 R 3.5The results demonstrate that streptococci B can be detected early usingan esterase enzymatic substrate since they exhibit a zero to very weakactivity at 18-24 h.

EXAMPLE 2 Detection of Streptococcus Agalactiae Using an EsteraseSubstrate and an α-glucosidase Substrate or Phosphatase Substrate

The protocol described above in Example 1 was repeated, with theexception that, at the same time as 0.3 g/l of the esterase substrateX-C8, 0.3 g/l of 6-chloro-3-indolyl-α-D-glucopyrano side (Rose-α-Glu),or 0.3 g/l of 6-chloro-3-indolyl phosphate (Rose-P), which give a pinkcoloration when they are used, was added.

The results are given in Table 2 below, in which the growth, thecoloration and the intensity are given, as in Example 1, and whereR=Pink/(Rose)/Red, PB=Pink-Brown, T=Turquoise, Gr=Green, Vi=Violet,B=Blue, GVi=Grey-Violet and GB=Grey-Blue.

TABLE 2 X-C8 + Strains Rose-alpha-Glu X-C8 + Rose-P (accession No.) T GCo I G Co I Streptococcus 18 1.3 R 3 1 R 3 agalactiae 24 1.3 R 3 1.7 R 4(7611003) >40 2 R 4 2 R 4 Streptococcus 18 0.2 R 2 0.5 R 3 agalactiae 240.3 R 2 0.5 R 3.5 (8709013) >40 1 R 4 1.7 R 4 Streptococcus 18 1 R 2 0.8R 3 agalactiae 24 1.7 PB 2.7 1.3 R 4 (7702055) >40 1.7 R 4 1.7 R 4Enterococcus 18 1.5 T 3 1.7 GB 3 faecium 24 1.7 T 3 1.7 B 3.5(7611005) >40 1.8 T 4 2 GVi 4 Staphylococcus 18 0.7 Gr 3 0.6 GVi 3.5epidermidis 24 1.3 GB 3.5 1.3 GVi 3.5 (7509009) >40 1.3 GB 3.5 1.5 GVi 4Staphylococcus 18 3 GVi 3 2 Vi 4 aureus 24 3 Vi 4 3 Vi 4 (9202070) >40 3Vi 4 3 Vi 4

This table demonstrates that the detection of the Streptococcusagalactiae strains is improved when a chromogenic esterase substrate isused in combination with another chromogenic enzymatic substrate, otherthan an esterase substrate, that can be used by the Streptococcusagalactiae strains.

EXAMPLE 3 Detection of Streptococcus Agalactiae Using an EsteraseSubstrate, a Phosphatase Substrate and a β-Cellobiosidase Substrate

The protocol described in Example 2 was repeated, using 0.3 g/l of X-C8and 0.2 g/l of Rose-P, with the exception that 0.08 g/l of5-bromo-4-chloro-3-indolyl-β-D-cellobioside (Cellobio) is also added atthe same time as the other substrates, along with 0.5 g/l of Na₂HPO₄ and0.5 g/l of K₂HPO₄, before autoclaving.

As control medium, a medium with only X-C8 and Rose-P was used.

The results are given in Table 3 below, in which the growth, thecoloration and the intensity are given, as in Example 1, and whereR=Pink/Rose)/Red, Ma=Mauve, Vi=Violet, B=Blue, GB=Grey-Blue and DP=DarkPurple.

TABLE 3 X-C8 + Rose-P + Strains Control Cellobio (accession No.) T G CoI G Co I Streptococcus 18 0.7 R 1.7 0.7 R 1.3 agalactiae 24 0.7 R 4 0.7R 3 (0101060) >40 1.5 R 4 1.5 R 4 Streptococcus 18 1.3 R 4 1 R 4agalactiae 24 1.5 R 4 1.5 R 4 (7701031) >40 1.5 R 4 1.5 R 4Streptococcus 18 1 R 2 1 R 2 agalactiae 24 1.5 R 4 1.5 R 4 (7702055) >401.7 R 4 1.7 R 4 Streptococcus 18 0.3 0.3 B 0.5 anginogus 24 0.5 R 0.10.4 B 1.3 (8507046) >40 1 R 2.3 1 B 2.7 Enterococcus 18 1.5 Ma 1.7 1.3 B3 faecium 24 1.7 Ma 3 1.5 GB 4 (0002043) >40 2 DP 4 2 DP 4

The results in Table 3 demonstrate an improvement in the specificity ofdetection of Streptococcus agalactiae compared with the other strainswhen three enzymatic substrates, including an esterase substrate, areused.

EXAMPLE 4 Detection of Streptococcus agalactiae Using an EsteraseSubstrate, a Phosphatase Substrate and an N-AcetylglucosaminidaseSubstrate

The protocol described in Example 3 was repeated, with the exceptionthat 0.4 g/l of 5-bromo-4-chloro-3-indolyl-β-N-acetylglucosaminide(X-NAGlu) was used in place of the Cellobio.

The control medium is identical to the medium tested, with the exceptionthat it does not contain any X-NAGlu.

The results are given in Table 4 below, in which the growth, thecoloration and the intensity are given, as in Example 1, and whereR=Pink/Red, B=Blue, GP=Grey-Pink and Mg=Magenta.

TABLE 4 X-C8 + Rose-P + Strains Control X-NAGlu (accession No.) T G Co IG Co I Streptococcus 18 0.5 R 3 0.5 R 2.3 agalactiae 24 1 R 4 1 R 3(7611003) >40 1.2 R 4 1.2 R 4 Streptococcus 18 0.5 R 2.7 0.5 R 2.7agalactiae 24 0.7 R 4 0.7 R 4 (7701031) >40 0.7 R 4 0.7 R 4 Enterobacter18 1.7 R 2.3 1.7 GP 2 clocae 24 2 R 3 2 B 3 (0010003) >40 2.5 B 4 3 B 4Enterococcus 18 0.5 GP 2 0.5 B 3 faecium 24 0.8 GP 2.7 0.8 B 3.5(0002043) >40 1 Mg 4 1 B 4

The results in this table demonstrate an improvement in the specificityof detection of Streptococcus agalactiae compared with the other strainswhen three enzymatic substrates, including an esterase substrate, areused.

EXAMPLE 5 Detection of Streptococcus agalactiae Using an EsteraseSubstrate, a Phosphatase Substrate and a β-Glucosidase Substrate

The protocol described in Example 4 was repeated, with the exceptionthat 0.08 g/l of 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside(X-β-Glu) and 0.3 g/l of5-bromo-4-chloro-3-indolyl-N-methyl-β-D-glucopyranoside (GreenA-β-Glu)were used in place of the X-NAGlu.

The control medium is identical to the medium tested, with the exceptionthat it does not contain any X-β-Glu or GreenA-β-Glu.

The results are given in Table 5 below, in which the growth, thecoloration and the intensity are given, as in Example 1, and whereR=Pink/Red, Ma=Mauve, Vi=Violet, B=Blue and GB=Grey-Blue.

TABLE 5 X-C8 + X-C8 + Rose-P + Rose-P + Strains Control X-β-GluGreenA-β-Glu (accession No.) T G Co I G Co I G Co I Streptococcus 18 0.40.3 0.2 agalactiae 24 0.5 R 0.5 0.5 R 0.3 0.4 R 0.3 (9001001) >40 1.7 R4 1.3 R 4 1.5 R 4 Streptococcus 18 1.3 R 4 1.3 R 4 1 R 4 agalactiae 241.5 R 4 1.5 R 4 1.5 R 4 (7701031) >40 1.5 R 4 1.5 R 4 1.5 R 4Streptococcus 18 1 R 2 1 R 2 1 R 2 agalactiae 24 1.5 R 4 1.5 R 4 1.3 R 4(7702055) >40 1.7 R 4 1.7 R 4 1.5 R 4 Streptococcus 18 0.3 0.3 B 3 0.3 B0.5 anginogus 24 0.5 R 0.1 0.4 B 4 0.4 B 2 (8507046) >40 1 R 2.3 1 B 4 1B 3.5 Enterococcus 18 1.5 Ma 1.7 1.5 B 4 1.5 GB 4 Faecium 24 1.7 Ma 31.5 B 4 1.7 GB 4 (0002043) >40 2 Vi 4 2 B 4 2 GB 4

The results obtained in Table 5 demonstrate an improvement in thespecificity of detection of Streptococcus agalactiae compared with theother strains when three enzymatic substrates, including an esterasesubstrate, are used.

EXAMPLE 6 Improvement in the Sensitivity of Detection by AddingPhosphate Solution

The protocol described in Example 1 was repeated, with the exceptionthat 0.3 g/l of Rose-P and also 0.5 g/l of Na₂HPO₄ and 0.5 g/l of K₂HPO₄were added at the same time as 0.3 g/l of the esterase substrate X-C8.

The same medium, but with no phosphate solution, was used as controlmedium.

The results are given in Table 6 below, in which the growth, thecoloration and the intensity are given, as in Example 1, where R=Pinkand Mg=Magenta.

TABLE 6 Medium with Strains Control phosphate solution (accession No.) TG Co I G Co I Streptococcus 18 1.3 R 3 1.3 Mg 3.5 agalactiae 24 1.7 Mg 41.7 Mg 4 (7611003) >40 1.8 Mg 4 1.8 Mg 4 Streptococcus 18 0.4 R 0.5 0.5Mg 3 agalactiae 24 0.5 Mg 4 0.7 Mg 3.5 (0101060) >40 1.5 Mg 4 1.5 Mg 4Streptococcus 18 1 Mg 3 1 R 3.5 agalactiae 24 1.5 Mg 4 1.5 Mg 4(7702055) >40 1.5 Mg 4 1.5 Mg 4

The results in this Table 6 demonstrate an improvement in the sharpnessof coloration from 18 h, or an increase in the expression of the S.agalactiae strains.

EXAMPLE 7 Comparison of the Sensitivity and the Specificity of Detectionof S. Agalactiae Using a Medium Containing an Esterase SubstrateAccording to the Invention and the Commercially Available Media

For this sensitivity and specificity study, a medium according to theinvention, prepared as described in Example 1, containing 0.3 g/l ofX-C8 and also: 0.2 g/l of Rose-P, 0.08 g/l of Cellobio, 0.5 g/l ofNa₂HPO₄, 0.5 g/l of K₂HPO₄, 0.012 g/l of aztreonam and 0.004 g/l ofamphotericin B, was used.

As medium for comparison, the Granada medium (ref. 10 077, BIOLYS,France) (Granada medium) was used.

69 strains of microorganisms, including 14 of Streptococcus agalactiae,were inoculated and left to incubate at 37° C. for up to 24 h and atambient temperature beyond this time. The colonies were visualized asdescribed above. The confirmation of the colonies suspected of beingcharacteristic of streptococcus B, i.e. appearing to be pink/red, wascarried out by means of an agglutination assay using the Slidex StreptoKit reagent according to the supplier's recommendations (bioMérieux,France). The non-characteristic colonies, i.e. the colonies that wereother than pink or that had the characteristic coloration but gave anegative response in the agglutination assay (false-positive strains),were identified by means of Galeries ID 32 Strep (bioMérieux, France).

The results are expressed as % of correct diagnosis relative to all thetests in terms of sensitivity and specificity, and are given in Table 7below, the % sensitivity corresponding to the number of true positivesdetected on the medium divided by the total number of true positives tobe detected (*100), and the % specificity corresponding to the number oftrue negatives detected on the medium divided by the total number oftrue negatives to be detected (*100).

TABLE 7 % sensitivity and specifity of detection of S. agalactiaeGranada medium Medium of the invention 18 h 24 h >40 h 18 h 24 h >40 hSensitivity without 50 50 50 79 79 93 enrichment Sensitivity with 50 5050 79 86 93 enrichment Specificity without 100 100 100 87 82 80enrichment Specificity with 100 100 100 89 93 82 enrichment

The results indicated in this table demonstrate the improvement in thesensitivity of detection of streptococci B (Streptococcus agalactiae)using the method of the invention. Moreover, they also demonstrate thatthe detection medium of the invention also has good specificity, whichspecificity is improved after enrichment due to a passage in Todd-Hewittbroth for 18-24 hours at 35-37° C. with or without 5% CO₂ beforeinoculation of the agar (see CDC (Center for Disease Control)recommendations, MMWR (Morbidity and Mortality Weekly Report), 16 Aug.2002, Vol. 51, No. RR-11).

EXAMPLE 8 Use of the Medium Based on Clinical Samples

For this study, the medium according to the invention, as prepared asdescribed above in Example 7, was used.

A total of 134 samples/swabs originating from vaginal or endocervicalspecimens from pregnant women were used in this study.

Each swab was emulsified in 1 ml of sterile physiological saline and 100μl of this solution were deposited, firstly, onto a Columbia agarcontaining 5% of horse blood, and, secondly, onto the medium used in themethod of the invention. Moreover, 100 μl of the above solution wereused to inoculate a Todd Hewitt broth. After incubation for 20 hours at37° C. and under aerobic conditions, the blood-agar and the medium ofthe invention were inoculated using the Todd Hewitt broth and thenincubated at 37° C. for 20 h under aerobic conditions.

The confirmation of the colonies suspected of being characteristic ofstreptococcus B, i.e. that appeared pink/red in color, was carried outby means of an agglutination assay using the Slidex Strepto Kit reagentaccording to the supplier's recommendations (bioMérieux, France).

Among the 134 samples, 112 were inoculated onto the agar media, firstly,directly from the suspension in physiological saline and, secondly,after enrichment in Todd Hewitt broth. The remaining 22 samples wereinoculated onto the agar media only directly from the suspension inphysiological saline.

The results, expressed as average percentage sensitivity andspecificity, are presented in Table 8 below.

TABLE 8 Columbia agar Invention agar Sensitivity 95 100 Specificity 9099.5

The results in Table 8 above show that the medium of the invention, usedwith clinical samples, makes it possible to improve the sensitivity andthe specificity of detection of Streptococcus agalactiae. Specifically,20/20 specimens containing Streptococcus agalactiae are detected on themedium of the invention, against 19 on the Columbia medium, and there isjust one false+result on the esterase medium, against 24 on the Columbiaagar. It can even be noted that the results are better than when themedium was tested with the laboratory strains.

What is claimed is:
 1. A reaction medium, comprising: (i) an esterasesubstrate that Streptococcus agalactiae is not capable of using at lessthan 18 hours after inoculation; (ii) at least one enzymatic substrateselected from β-cellobiosidase substrates, N-acetylglucosaminidasesubstrates, and β-glucosidase substrates; and (iii) a phosphatasesubstrate.
 2. The reaction medium according to claim 1, wherein the atleast one enzymatic substrate is a β-cellobiosidase substrate.
 3. Thereaction medium according to claim 1, wherein the at least one enzymaticsubstrate is an N-acetyl-glucosaminidase substrate.
 4. The reactionmedium according to claim 1, wherein the at least one enzymaticsubstrate is a β-glucosidase substrate.
 5. The reaction medium accordingto claim 4, wherein the β-glucosidase substrate is an indoxyl substrate.6. The reaction medium according to claim 1, wherein the esterasesubstrate is selected from derivatives of indoxyl octanoate, indoxylnonanoate, and indoxyl decanoate.
 7. The reaction medium according toclaim 1, wherein a concentration of each enzymatic substrate is between10 and 2000 mg/l.
 8. The reaction medium according to claim 1, furthercomprising a phosphate solution.
 9. The reaction medium according toclaim 8, wherein the phosphate solution is Na₂HPO₄ or K₂HPO₄.
 10. Thereaction medium according to claim 1, further comprising a mixture ofinhibitors for inhibiting or limiting growth of unwanted strains. 11.The reaction medium according to claim 10, wherein the inhibitors areantibiotics.
 12. The reaction medium according to claim 11, wherein theantibiotics are aztreonam and amphotericin B.